Although therapy with adult bone marrow (BM)-derived unfractionated mesenchymal stem cells (MSCs) improves cardiac function, remodeling, and perfusion after MI, the extents of MSC-induced angiogenesis, myogenesis, and antiapoptotic and `paracrine effects'have been inconsistent in studies from different laboratories. These apparent discrepancies might have stemmed from the heterogeneous nature of unfractionated `MSCs', and the use of dissimilar expansion protocols in different laboratories. Our fundamental hypothesis is that a primitive and homogenous MSC subpopulation isolated on the basis of surface antigen expression will exhibit greater endothelial and cardiomyogenic differentiation potential, and secrete greater amounts of cardioprotective growth factors/cytokines;and that optimization of medium is critical for the expansion of MSC subpopulation specifically intended for cardiac repair without significant changes in antigenic phenotype, gene expression, differentiation potential, and secretory profile. Accordingly, Aim 1 will thoroughly examine whether compared with unfractionated MSCs, an antigenically-defined MSC subpopulation exhibits a more primitive phenotype, expresses endothelial/cardiomyogenic lineage markers, and undergoes greater endothelial/cardiomyogenic differentiation. Expression of markers of pluripotency and lineage commitment will be compared using real-time RT-PCR in unfractionated MSCs and MSC subpopulations isolated by flow cytometry based on the expression of CD45, c-kit, Flk-1, Sca-1, CD90, and CD105. Endothelial and cardiomyogenic differentiation will be rigorously quantitated (morphology, immunocytochemistry, confocal microscopy). To predict in vivo efficacy, differentiation (and secretory profile in Aim 2) will be assessed in culture conditions that simulate the in vivo milieu. Aim 2 will examine whether an antigenically-defined MSC subpopulation secretes greater amounts of cardioprotective growth factors/cytokines, and whether gene expression profiles correlate with secretory profiles. Aim 3 will conclusively establish the optimal medium composition for the expansion of select MSC subpopulation(s) with no or minimal alterations in surface antigen expression, gene expression, differentiation potential, and secretory abilities. Four primary media (DMEM, IMDM, 1-MEM, and Mesencult) and five factors (LIF, IGF-1, FGF-2, PDGF-B, and Wnt3a) will be examined. Antigen and gene expression, differentiation, and secreted factors will be examined during serial passages. In summary, this exploratory/developmental proposal aims to identify a novel MSC subpopulation most suitable for cardiac repair, and to establish the optimal medium that will expand these cells without significant changes in surface antigen expression/gene expression/differentiation potential/secretory attributes for future cardiac repair in vivo. The results of the proposed investigations will not only provide novel insights into MSC biology, but may also identify a cellular population ideally-suited for cardiac repair in patients with ischemic heart disease and post-MI heart failure. PUBLIC HEALTH RELEVANCE: Ischemic heart disease is the single most prevalent cause of death and morbidity in all Western countries. Despite significant advances in medical treatments, the prognosis for patients with large heart attacks is poor with eventual progression to end-stage heart failure and death. Although recent studies indicate that therapy with adult primitive cells can repair dead heart muscle, the results have been variable, and the mechanisms remain unclear. Thus, the identification of an ideal cell for heart repair, a major goal of this exploratory proposal would have enormous therapeutic implications for patients with ischemic heart disease and heart failure. The optimization of culture medium for expansion of this cell population will help generate a large number of cells for potential use in vivo. The results of the proposed studies will therefore be highly relevant for improving public health, and improving the length and quality of life of millions of patients with ischemic heart disease and heart failure.